At present, the most common technique for the detection of polynucleotide sequences is hybridization using oligonucleotide or polynucleotide probes. For this technique, the target nucleic acid is usually bound, irreversibly, to a solid support such as cellulose or nylon. The labelled probe is then added in solution under hybridizing conditions and if there is sequence homology between the probe and the target nucleic acid, the probe will form a hybrid molecule with the target polynucleotide. This hybrid can be detected in a variety of ways such as by radiolabelling or biotin labelling. The disadvantages of this technique are, firstly, that it requires considerable operator expertise, secondly, the technique is lengthy and time-consuming and thirdly, cannot be easily automated. Often the entire procedure can take more than 48 hours.
The liquid-solid methods normally employed for detecting specific nucleic acids in samples include Southern blot, Northern blot and dot blot hybridizations. These methods are slow, inefficient, technically demanding and are not easily automated. The Southern and Northern blot protocols have the further disadvantage of inefficient transfer of nucleic acid from gel to paper. Other groups have used liquid-solid hybridizations in which a capture probe is bound to a solid support and the DNA sequence of interest in the sample becomes bound to the capture probe. An example of this is in Australian Patent specification no. AU-70152/87 which describes using at least two oligonucleotide probes, which are complementary to mutually exclusive regions of the same target nucleic acid in a liquid hybridization format to anneal the probes to the target, if it is present, and detecting the presence of the target by immobilisation of the two-probe target sandwich by one of the probes and subsequent detection of the other probe. However, this method requires a second, detector probe to hybridise to the DNA sequence of interest. This step reduces the specificity of the assay and subsequently increases background. The present invention overcomes this problem by involving only one probe which acts both as capture probe and as detector probe.
In contrast to liquid-solid hybridization, liquid-liquid hybridization has very rapid kinetics and improved sensitivity due to greater access of the probe to the target sequence. For example, Gene-Probe, Inc., uses a liquid hybridization hydroxapatite method to detect DNA sequences. The main disadvantage of this system is that it relies on adsorptive discrimination of double-stranded DNA from single-stranded DNA sequences rather than sequence-specific separation of hybrid from excess probe. The present invention overcomes these disadvantages by allowing the nucleic acid hybridizations to occur in solution followed by the removal of the "hybrid" molecules onto a solid support matrix. Another potential advantage of liquid hybridization is that a generalised solid support can work for a multitude of targets if the support-binding probes are labelled with the same capture molecule.
Several cases exist (Australian Patent specification nos. AU-A-70152/87, AU-A-26755/88, AU-A-53105/86, AU-B-89575/82 and AU-A-80097/87) which use a combination of two oligonucleotide probes to detect specific nucleic acid sequences in a sample. All these require the use of two short sequences of DNA on the target. These sequences must both be conserved in all possible target, must be mutually exclusive and non-overlapping and must have a similar G+C ratio to enable both probes to hybridize to their complementary sequence under the same conditions.
There is related background art concerning the use of a capture probe to detect the nucleic acid sequence of interest and to remove it from solution by binding the hybrid to a solid support matrix (Australian Patent specification nos. AU-A-70152/87, AU-A-53105/86, AU-A-21781/88, AU-A-69724/87, AU-A-14005/88). However, these techniques use separate capture and detector probes, resulting in a number of disadvantages as detailed above. The present invention overcomes these problems by using a single capture/detector probe system.
There are many examples available of attaching non-radioactive reporter molecules to DNA, to enable the detection of specific hybrids. However, when biotin is incorporated into the DNA molecule for detection, even though several biotin molecules may be incorporated per target molecule (thereby increasing the sensitivity of detection) the mechanism of visualising the incorporated biotin is complex and time consuming.
By contrast, the incorporation of other non-radioactive reporter molecules (such as fluorescent, luminescent, chemiluminescent molecules) enables rapid and simple detection of the target sequence. However, the present art only enables a single reporter molecule to be attached to each target sequence. This fact reduces the overall sensitivity of the final assay. The present invention overcomes both these problems at the one time by using a chemiluminescent detection system for simple, rapid detection and also by incorporating several detector molecules into each target, thereby significantly increasing assay sensitivity.
There is, therefore, a demand for a simple method which utilises the rapid kinetics of liquid hybridization, which only requires a single probe for analysis, and which results in stable hybrids thereby allowing the unhybridized material to be easily removed from the sequences to be detected. Accordingly, the present invention provides a liquid hybridization system in which a single probe hybridizes to the sequence of interest and is then covalently extended to produce a stable hybrid. This hybrid is then captured on to a support matrix and subsequently washed to removed unhybridized material. The system described by the present invention is simple, rapid, sensitive, can be read visually or on a simple plate reader, and may be readily automated.
In this area of nucleic acid hybridization there is a need to detect two broad types of diseases: infectious and genetic. In relation to infectious diseases, a number of DNA based systems have been described to detect diseases caused by bacteria such as Salmonella, Neisseria, parasitic organisms such as Chlamydia and Rickettsiae, viruses such as hepatitis B and protozoa such as Plasmodium. However, all of these suffer one or more of the disadvantages listed above.
In relation to genetic diseases which are characterised by a mutation (deletion, insertion, point mutation or translocation), the technology is less well developed. These types of diseases are currently diagnosed either by using restriction fragment length polymorphism (RFLP) analysis or by precise hybridization of short oligonucleotide probes. RFLP detection requires that a restriction enzyme site is altered by the mutation and this is not always the case. In addition, RFLP analysis requires the use of Southern blot hybridization for detection. The use of short oligonucleotide probes to detect point mutations also has several serious disadvantages. In particular, the hybridization conditions must be precise to ensure that a single base-pair mismatch does not result in hybridization. In practice, salt concentration and temperature determine the specificity of the hybridization conditions and these are not easily controlled to the required preciseness. The present invention overcomes the need for Southern hybridization analysis and for precise control of hybridization conditions. It achieves this by the specific primer probe hybridizing to a constant section of the gene adjacent to the mutation and allowing the enzyme, a DNA polymerase, to extend the DNA chain up to and including the nucleotide or base mutation. By manipulation of the dideoxynucleotide added to the polymerase reaction all of the possible nucleotide changes can be detected.
Detection of both infectious and genetic diseases requires the incorporation of some type of labelled molecule into the system. Various radioactive isotopes or radioactivity labelled compounds may be used as labels. However, radioactive labels have several disadvantages, including; (i) hazardous, (ii) expensive, (iii) limited shelf life, (iv) require expensive equipment for measuring the signal generated. More recently, a range of non-radioactive substances have been used to detect DNA molecules. Examples of non-radioactive labels are fluorescent, luminescent, chemiluminescent, enzymatic or immunological compounds. Labels based on the affinity of biotin and avidin or streptavidin, lanthanide chelates, lectins and proteins may also be used. The preferred detection means would be by spectroscopy or photochemistry or by the formation of a detectable complex between the label moiety and the polypeptide, lectin, or antibody linked to an entity capable of generating a detectable change, including enzymes such as; alkaline phosphatase or horseradish peroxidase.
However, what is lacking in the current technology is a single system which encompasses: (i) a rapid means of detecting a polynucleotide sequence, (ii) which is sufficiently sensitive to detect low numbers of the target sequence in the sample and (iii) which is non-radioactive. At present, no single system satisfies all these requirements.
As a final aspect, the need to detect specific polynucleotide sequences in a sample requires the organisation of all steps either; (i) into a simple kit format, or (ii) into an automated device. Whereas both, kits and automated machines, are available for detecting proteins by way of antibodies, no systems are yet available which simply, rapidly and inexpensively detect specific polynucleotide sequences in a sample.